Cell Reprogramming & Therapeutics LLC will develop new cellular reprogramming technology to produce cell therapeutic product for the treatment of Parkinson's disease (PD). Advances in cell reprogramming technologies to generate patient-specific cells of a desired type will revolutionize the field of regenerative medicine. Over the lat decades several cell reprogramming methods such as nuclear transfer, cell fusion and transfection or transduction with pluripotent factors have been developed. However, the majority of these technologies require the exposure of cell nuclei to reprogramming large molecules via transfection, transduction, cell fusion or nuclear transfer. This raises several technical, safety and ethical issues. Chemical genetics is an alternative approach for cell reprogramming that uses small, cell membrane penetrable substances to regulate multiple cellular processes including cell plasticity. The main advantages of this approach over the above-mentioned technologies are that the biological effects of small molecules are typically rapid, reversible and dose-dependent, allowing precise control over specific outcomes by fine-tuning their concentrations and combinations. Recently, using chemical genetics approach (the combination of small molecules that are involved in the regulation of chromatin structure and function and specific cell signaling pathways), we have been able to generate neuronal cells from human mesenchymal stem cells (hMSCs) that expressed mature dopaminergic (DA) markers, released dopamine, exhibited electrophysiological properties of maturing neurons, and formed synapses. The goal of the proposed Phase I studies is the optimization of this cell reprogramming technology to increase the production of engraftable (FOX2A+/TH+/Nurr1+) midbrain DA neurons and elucidation of the therapeutic effects of these specialized cells in an animal model of PD. Phase II studies will focus on clinical grade manufacturing of these DA cells and testing their therapeutic effect in several preclinical pathogenic and etiologic animal models of PD. Commercial and clinically compatible research products emerging from Phase I/II work include a DA neuron differentiation kit and a technology for large-scale clinical grade production of DA neurons. The ultimate post-Phase II product will be the production of clinically relevant DA neurons for PD therapy. To achieve these goals, the Phase I aims include: Specific Aim 1 will test the hypothesis that the efficiency of DA specification and maturation can be further improved by addition of specific cell signaling modulators and growth factors to our recently developed neural induction protocol at a certain time and in a specific order. Immunocytochemistry and RT-PCR will be used to evaluate the expression of markers specific to immature and mature dopaminergic neurons. Dopamine release will be assessed by ELISA. Specific Aim 2 will test the hypothesis that DA cells transplanted into the 6- hydroxydopamine (6-OHDA) mice and rat models of PD, will survive, integrate and will promote restoration of amphetamine-induced rotation behavior and will show improvements in tests of forelimb use and akinesia.